Author

Date of Award

Document Type

Degree Name

Administrative Home Department

Department of Chemistry

Advisor 1

Haiying Liu

Committee Member 1

Christo Z. Christov

Committee Member 2

Tarun K. Dam

Committee Member 3

Lei Pan

Abstract

Lysosome is an acidic membrane-bound organelle containing more than 70 hydrolytic enzymes that breaks down different biological macromolecules. Substantial lysosomal pH disruption can cause lysosome malfunction and consequently lead to lysosomal storage disease. Therefore, it is essential to precisely monitor lysosomal pH changes in order to explore cellular functions and get insightful understanding of physiological and pathological processes. Fluorescence imaging based on fluorescent probes is a powerful technique to monitor lysosomal pH changes because of advantageous features including operational simplicity, high sensitivity, non-invasive approach, and high spatial resolution. However, most of the reported lysosomal pH probes are based on Stokes-shift fluorescence with lower energy emission under higher energy excitation, and exhibit less than 600 nm absorption and emission wavelengths, which causes cellular and tissue photodamage and contains biological fluorescence background. In order to avoid these issues, we developed near-infrared fluorescent probes based on single-photon anti-Stokes fluorescence with near-infrared excitations and emissions. We significantly improved biocompatibility and water-solubility of fluorescent probes by introducing mannose residues to the fluorophores through oligo(ethylene glycol) tethered spacers for sensitive detection of lysosomal pH changes in two near-infrared channels. In order to take advantage of ratiometric and near-infrared imaging to overcome systematic errors of intensity-based fluorescent probes caused by probe concentration variation and uneven distribution, temperature, solvent polarity, and excitation light fluctuation, we developed ratiometric near-infrared fluorescent probes for ratiometric detection of lysosomal pH changes by introducing tetraphenylethene (TPE) dyes to hemicyanine dyes. Gradual lysosomal pH decreases result in gradual increases of hemicyanine fluorescence, and corresponding concomitant decreases of TPE fluorescence. The probes allow for development of various ratiometric near-infrared fluorescent probes for quantitative and comparative reliable analyses of cations, reactive nitrogen, oxygen and sulfur species by conjugating various biosensing groups into the near-infrared hemicyanine moieties.